Lysine acetylation and deacetylation play important roles in the modulation of chromatin topology and the regulation of gene transcription. Lysine deacetylases and histone deacetylases in particular are proven drug targets for cancer and also potential targets for neurological diseases. A commonly used assay for HDAC activity is a trypsin coupled fluorogenic assay. This indirect endpoint assay is simple and applicable for high throughput screening. However, it is limited in its ability to continuously monitor enzyme activity due to protein's stability in the presence of trypsin. In addition, trypsin inhibitors may impair the assay results. Recently, Caliper microfluidic lab-on-a-chip technology has been used to measure HDAC activity and characterize HDAC inhibitors. This assay directly follows the separated fluorophore-labeled substrate and product using FAM labeled acetylated peptide. Advantages of this direct assay include its ability to continuously monitor enzyme activity and the ability to determine enzyme activity in the absence of trypsin or other proteases which may degrade protein components within the assay. Interference from fluorescent compounds is minimized in screening. This assay is currently limited for a few HDACs such as HDAC3 and 6 due to the lack of efficient HDAC substrates for other HDAC isoforms including HDAC1 and 2. As such, substrates used to determine the activity of all HDAC isoforms using the microfluidic lab-on-chip technology is needed.